The present invention generally pertains to the field of electrophoresis, and specifically to an apparatus providing improved circulation of the liquid buffer solutions used in electrophoresis.
Electrophoresis devices are typically used for the separation of charged biological molecules, such as proteins and nucleic acids, by differential migration of the molecular constituents through a semi-solid matrix. The migration is caused by an electrical field induced between two electrodes connected to an external DC power supply. The rates of migration of individual macromolecules is determined by their mass and net electrical charge.
The observation that electrically charged biopolymers will migrate in response to an electrical field has led to the development of various electrophoresis techniques. In gel electrophoresis, matrices such as agarose, polyacrlyamide or starch, when placed in a liquid buffer solution, are used as a medium through which mixtures of macromolecules will migrate. The sample mixture is applied to one end of the matrix plate and current is applied across the matrix. The molecules in the sample migrate to discrete zones, or bands, based on their mass and charge.
Following electrophoresis, a wide variety of both direct and indirect techniques are used to visualize the macromolecules either within the electrophoresis matrix or following transfer to a solid support. Both analytical and preparative and qualitative and quantitative methods are known and used. Many of these methods are described in ELECTROPHORESIS Theory, Techniques, and Biochemical and Clinical Applications, 2nd edition, A. T. Andrews, Oxford University Press, 1986; Current Protocols in Molecular Biology, ed. Ausubel, F. M., et al, Wiley Interscience, 1990; and GEL ELECTROPHORESIS OF NUCLEIC ACIDS: A Practical Approach, ed. Rickwood, D. and Hames, B. D., IRL Press, Oxford, England, 1990.
Electrophoresis, and specifically gel electrophoresis, is widely used in clinical and forensic laboratories to separate isoproteins obtained from blood and other body fluids in order to diagnose specific disease states, or to identify the source of an evidence sample. It is also used to separate deoxyribonucleic acid (DNA) molecules for the purposes of medical genetics and individual identification. In research laboratories, electrophoresis forms the backbone technique of many life science fields, such as molecular biology and genetics and protein biochemistry.
In gel electrophoresis of macromolecules such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), circulation of the liquid buffer solution is necessary for molecular migration to occur. Circulating the buffer overcomes imbalances in chemical and thermal conditions that develop during the course of an electrophoeretic separation and that lead to distortions of the migration pattern in the gel. These distortions occur because electrophoretic separation of nucleic acids may take as many as twelve to thirty hours.
This lengthy electrophoresis time period causes drops in the current flow that can exceed 50% at constant voltage. This current drop is caused by the deposition of oppositely charged ions at the anode and cathode of the electrophoresis chamber. During electrophoresis, protons (H.sup.+) are formed at the anode and hydroxide ions (HO.sup.-) at the cathode as a result of the electrolysis of water. This effect can be quantified by measuring the pH of the liquid buffer solutions in the buffer reservoirs at the anodic and cathodic ends of the electrophoresis chamber. Commonly used buffer solutions have a pH of 8, but following a 16 hour electrophoresis run, the pH can be approximately 10 at the anode and 6 at the cathode. This pH grandient causes slowing of the run and, depending on the relative amounts of sample loaded in a particular lane, can lead to local ion effects that selectively retard migration in the region of a particular sample.
Another factor that can affect the migration patterns is uneven dissipation of the heat that is generated during the course of the electrophoresis run. This heat is a product of the resistance associated with the current passing through the electrophoresis matrix. The above adverse effects can be overcome by circulating the liquid buffer solution between the two buffer reservoirs and over the electrophoresis matrix.
Currently, solution circulation is accomplished by the use of external peristaltic or other non-conducting pumps that are connected by plastic tubing to the two buffer reservoirs. These pumps experience wear and tear and are prone to break down. Also, mechanical pumps require a separate energy supply, are cumbersome to install, and may contaminate the buffer solution. U.S. Pat. 4,702,814 describes non-mechanical circulation of electrophoresis buffer solution by movement of gas bubbles from one electrophoresis buffer receptacle to the other along an inclined conduit. This gas bubble migration concomittantly carries and propels buffer fluid from one receptacle to the other as an inherent part of the bubble migration process.